Student Instructions for the Purification of Genomic DNA from Escherichia coli

The purification of DNA is an important laboratory technique in a modern biochemistry

laboratory. The purified DNA (either genomic or plasmid) is used in several applications. For example,

one may wish to conduct biophysical experiments with the DNA (e.g., thermal denaturation studies), or

one may wish to use the DNA in cloning, PCR, or other biotechnological experiments. In this

laboratory investigation, you will purify genomic DNA from E. coli and ultimately use this purified

genomic DNA as a template in a subsequent PCR. (The present instructions will be limited to the

purification of genomic DNA, however.)

To purify genomic DNA you need to have a source (yours is E. coli), a means to lyse the cells,

and a strategy to (i) protect the genomic DNA from degradation, (ii) minimize shearing of the genomic

DNA, and (iii) separate the genomic DNA from other contaminating biomolecules in the bacterial cells.

To lyse the cells, you will be using a combination of a laundry detergent (Tide Free 2X Ultra), which

contains enzymes to help degrade unwanted proteins, polysaccharides, and lipids, and n-butanol, which

has limited solubility in water and thus forms a separate (organic) phase from the aqueous phase. You

also will be using 2-propanol and ethanol to precipitate the genomic DNA, which will mostly have

been freed of contaminating proteins during the step in which n-butanol is used.

The reagents listed in the protocol below have been chosen because their use represents a

greener approach than more traditional methods of purifying genomic DNA, which often involve the

use of chloroform, isoamylalcohol, and phenol. It is important to remember, however, that although n-

butanol, 2-propanol, and ethanol have significantly fewer environmental hazards associated with their

use than do solvents such as chloroform, isoamylalcohol, and phenol, they are still alcohols and thus

they should be used with caution as discussed in the Hazards section below.

The protocol that you will be using is listed on the following page so that you can see all of the

instructions contained on one page. As part of the pre-lab, however, you will need to create a separation

scheme in which you list the bare-minimum instructions in a flow-chart format.

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Materials

Micropipettors, sterile micropipettor tips, 1.5 mL microcentrifuge tubes, microcentrifuge

1 mL of an overnight (O/N) culture of E. coli K-12

Tide Free 2X Ultra

n-butanol (straight or water-saturated)

Vortex mixer

2-propanol

70% (v/v) ethanol

Drawn-out Pasteur Pipette

0.1 mg/mL ribonuclease A (RNase A) in autoclaved dH2O

Hazards

Observe all standard laboratory precautions (e.g., wear safety goggles at all times; wear gloves

when handling the bacterial culture; treat biohazard waste with bleach; place pipette tips and

microcentrifuge tubes that were in contact with the bacterial culture in biohazard bags and autoclave).

Use care when handling the drawn-out Pasteur pipettes as the ends are very sharp. The solvents n-

butanol, 2-propanol, and ethanol are flammable; consult the MSDS of each of these solvents prior to

starting the laboratory. All waste will be properly disposed of in the appropriate containers.

Protocol

1) Aliquot 1 1.4 mL of an overnight culture of E. coli into a 1.5 mL microcentrifuge tube, and
spin at high speed (~ 16,000 g) on a benchtop microcentrifuge for 1 minute. Note that for
this and for all subsequent spins the hinges connecting the caps to the tubes should be
oriented outwards so that the location of the pellet will be known see blackboard.

2) Remove all but 0.4 mL of supernatant from the tube, recap the tube, and thoroughly
resuspend the cells with a vortex mixer. The removed supernatant should be added to a
bleach-containing beaker.

3) Add 5 L of Tide Free 2X Ultra to each tube and vortex briefly to mix (about three, 2-second
pulses).

4) Incubate the tubes at 37 C for 10 min.

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 5) Add 750 L of n-butanol (straight or water-saturated) to each tube; vortex briefly to mix
(two, 5-second pulses).

6) Spin at top speed for 5 min.

7) Carefully lower a clean 10 200 L pipette tip through the organic layer and into the lower
aqueous layer. You likely will have to push aside a film of material at the interface of the
two layers. Slowly withdraw 200 L of the aqueous layer, taking care not to disturb the pellet
at the bottom of the tube or to siphon any of the precipitated material at the interface, and
transfer to a new, sterile microcentrifuge tube see blackboard.

8) Add 700 L of 2-propanol to each of the 200-L aqueous samples. Gently invert 5 times to
mix. You likely can see the precipitate of genomic DNA form upon initial mixing of the
sample.

9) Spin at top speed for 5 min.

10) Carefully decant the supernatant into the waste beaker and drain tubes on a Kim-wipe. You
likely will see a slightly yellow pellet at this point. You should know where to expect the
pellet because of the manner in which you oriented your tubes in the microcentrifuge see
#1 above and blackboard.

11) Add 700 L of 70% EtOH to your tubes containing the pellets. Gently invert 5 10 times to
mix.

12) Spin at top speed for 2 min.

13) Carefully remove supernatant with a drawn-out Pasteur pipette. Be very careful not to disturb
the pellet, but make sure to remove all visible traces of liquid from the sides of the tube see
blackboard.

14) Allow the open tube to dry for 5 min. If you do not see any traces of liquid, proceed to the
next step.

15) To each tube, add 50 L of a solution containing 0.1 mg/mL RNase A in autoclaved dH2O.
Incubate at 60 C for 60 min. Midway through the incubation, remove tubes and gently tap
the sides to dislodge any tightly adhering pellets. Substitute 8 mM NaOH in place of RNase
A if desired.

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Results and Discussion

You will have to wait until the following laboratory period to check the results of your

purification. At this time, you will analyze your purified genomic DNA by agarose gel electrophoresis.

When the gel analysis is complete, you can finish writing your laboratory report. Some considerations

to address in your report are listed in the following questions. Some of these questions can be answered

during the pre-lab; other questions will have to await the results of the gel analysis. You likely will

have to consult journal articles, methodology texts, and the Internet to answer some of the questions.

1. What role does the Tide Free 2X Ultra play in the lysis of the cells? Tide contains enzymes, but are

the enzymes the sole reason that Tide is used? (Hint: think about the structure of E. coli cells e.g.,

their Gram staining classification and what else in the Tide may have helped to lyse the cells.)

2. What components of the cells are dissolved in the n-butanol layer?

3. Why is the vortex mixing at step 5 of the protocol so brief? Would a longer duration improve the

yield of genomic DNA?

4. What step(s) likely helped prevent the degradation of the genomic DNA by deoxyribonucleases

(DNases)?

5. What components of the cells formed the film at the interface between the organic and aqueous

layers?

6. What causes the DNA to precipitate when 2-propanol is added at step 8 of the protocol?

7. Why is 70% ethanol used in step 11 of the protocol?

8. Why does one have to be so careful to remove all visible traces of liquid in steps 13 and 14 of the

protocol?

9. At step 15 of the protocol, the genomic DNA is rehydrated by adding an aqueous solution that

contains the enzyme RNase A. What is the purpose of using RNase A? Should you be concerned that

the incubation temperature of 60 C will inactivate the enzyme?

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10. Step 15 of the protocol states that 8 mM NaOH can be used in place of RNase A. What is the

justification of this statement? (Hint: think about the chemical differences between DNA and RNA.)

11. Is the appearance of the genomic DNA in the gel consistent with your expectations? In other words,

were there any surprises regarding the appearance of the DNA in the gel?

12. Other than checking the integrity of the DNA via gel electrophoresis, what other method(s) could

you use to test the purity of your isolated genomic DNA? (Hint: think about the max of proteins versus

the max of nucleic acids. Research to see if it is feasible to use the ratio of the absorbance at the max of

nucleic acids to that of the absorbance at the max of proteins as an indication of purity. What step(s) in

the above protocol would have to be altered to use such a method?

Literature Cited

1. Von Hippel, P. H.; Wong, K. Y. J. Biol. Chem. 1965, 240, 39093923.

2. Rex, J. H. Focus 2000, 22, 2627.
3. Boyer, R. Biochemistry Laboratory: Modern Theory and Techniques, 1st ed.; Benjamin
Cummings: San Francisco, 2006; p 297.
4. Blattner, F. R.; Plunkett, G. III,; Bloch, C. A.; Perna, N. T.; Burland, V.; Riley, M.;
Collado-Vides, J.; Glasner, J. D.; Rode, C. K.; Mayhew, G. F.; Gregor, J.; Davis, N.
W.; Kirkpatrick, H. A.; Goeden, M. A.; Rose, D. J.; Mau, B.; Shao, Y. Science 1997,
277, 14531474.
5. Gssow, D.; Clackson, T. Nucleic Acids Res. 1989, 17, 4000.
6. Sandhu, G. S., Precup, J. W., and Kline, B. C. BioTechniques 1989, 7, 689670.